Toad skin ointment for relieving swelling and pain as well as preparation method and application thereof

Abstract

The invention provides toad skin ointment for relieving swelling and pain as well as a preparation method and application thereof, and belongs to the field of medicines. The toad skin ointment is prepared from the following components in parts by mass: 2 to 3 parts of toad skin extract, 11 to 13 parts of vaseline, 11 to 13 parts of wool fat, 2 to 3 parts of liquid paraffin, 0.5 to 0.6 part of glycerol, 0.3 to 0.5 part of peppermint oil and 0.1 to 0.3 part of oil-soluble azone. The invention further provides a preparation method and application of the toad skin ointment. The toad skin ointment prepared by the invention has a remarkable inhibition effect on inflammation, central pain and peripheral pain, is non-irritant to skin, does not cause skin edema and erythema response, and is high in safety.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a toad skin ointment for reducing swelling and relieving pain, and a preparation method and application thereof. Background Art

[0002] Toad skin, also known as toad skin, dried toad skin, and clam skin, is the dried whole skin of the Chinese giant toad Bufo bufogargarizansCantor or the black-rimmed toad Bufo melanosticus Schneider. It has the effects of promoting diuresis and reducing swelling, relieving pain and eliminating malnutrition, clearing away heat and detoxifying, and can be used to treat swelling, edema, malignant sores, carbuncles and other diseases. Modern pharmacology shows that toad skin has anti-tumor, anti-inflammatory, antibacterial, and analgesic effects. Toad skin is widely used in clinical practice, especially Huachansu capsules and Huachansu injections made from dried toad skin as the main raw material, which have obvious anti-tumor effects. In addition, Huachansu can relieve various cancer-related pains by inhibiting tumor-related inflammatory reactions, greatly improving the quality of life of patients with middle and late stage cancer. However, due to the complex chemical composition of toad skin, many mechanisms of action are still unclear, and the toxic and side effects of internal use are large. At present, there are few studies on toad skin external preparations. Traditional toad skin external application is mostly fresh toad skin external application, or dry toad skin is directly applied externally after being powdered. The dosage is difficult to control during use, and it may even irritate the skin, posing a safety hazard. Therefore, preparing a toad skin external preparation with high safety and stable efficacy is of great significance to the development and utilization of toad skin. Summary of the invention

[0003] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a toad skin ointment for reducing swelling and relieving pain, and a preparation method and application thereof, which has a significant inhibitory effect on inflammation, central and peripheral pain, is non-irritating to the skin, does not cause skin edema or erythema reaction, and has high safety.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a toad skin ointment for reducing swelling and relieving pain. The ointment comprises the following components by weight: 2-3 parts of toad skin extract, 11-13 parts of vaseline, 11-13 parts of lanolin, 2-3 parts of liquid paraffin, 0.5-0.6 parts of glycerol, 0.3-0.5 parts of peppermint oil and 0.1-0.3 parts of oil-soluble azone.

[0006] Preferably, the toad skin extract is an extract of the dried whole skin of Bufo bufo gargarizans.

[0007] Preferably, the preparation method of the toad skin extract comprises the following steps: mixing the toad skin with ethanol, heating and refluxing for extraction, centrifuging the extract to obtain the supernatant, concentrating until there is no alcohol taste, and then freeze-drying to obtain toad skin freeze-dried powder.

[0008] Preferably, the solid-liquid ratio of the toad skin to ethanol is 1 g:15-25 mL; the ethanol is a 70-90% ethanol solution; the heating reflux extraction time is 2-3 h; and the heating reflux extraction times is 1-2 times.

[0009] Preferably, the centrifugal acceleration is 1700-1800×g, the time is 15-20 min; and the concentration temperature is 50-60°C.

[0010] Preferably, the freeze-drying temperature is -10 to -20°C, the vacuum degree is 150 to 300 Pa, and the time is 12 to 24 hours.

[0011] The invention also provides a preparation method of the toad skin ointment, comprising the following steps: dissolving vaseline, lanolin and liquid paraffin, mixing, adding glycerin and toad skin extract, mixing evenly, adding peppermint oil and oil-soluble azone, mixing evenly, and cooling to obtain the toad skin ointment.

[0012] Preferably, the dissolving is performed by heating in a water bath at 60-70°C.

[0013] The present invention also provides application of the toad skin ointment or the preparation method in preparing anti-swelling and analgesic drugs.

[0014] The present invention also provides application of the toad skin ointment or the preparation method in preparing anti-inflammatory drugs.

[0015] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0016] The toad skin ointment of the present invention has a significant inhibitory effect on inflammation, central and peripheral pain. The toad skin ointment can significantly reduce the expression content of IL-6, TNF-α, and IL-1β in serum and inflammatory tissue (P<0.01), and exerts an anti-inflammatory effect after skin administration; it can significantly increase (P<0.01) the β-EP content in serum and brain tissue, reduce the PGE2 content, and exert an analgesic effect after skin administration.

[0017] The toad skin ointment of the invention is non-irritating to the skin, does not cause skin edema or erythema reaction, and has high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Specificity chromatogram;

[0019] Figure 2 :The effect of toad skin ointment on mouse ear swelling model;

[0020] Figure 3 :The effect of toad skin ointment on carrageenan-induced foot swelling model in rats;

[0021] Figure 4 : TNF-α, IL-6, and IL-1β protein levels in rat serum and inflammatory tissues;

[0022] Figure 5 : Relative expression of TNF-α, IL-6, and IL-1β in inflammatory tissues of rats;

[0023] Figure 6 : β-EP and PGE2 protein levels in mouse serum and inflammatory tissues;

[0024] Figure 7 : Relative expression levels of POMC and PGE2 in mouse brain tissue;

[0025] Figure 8 :Photos of rats in single-dose skin irritation experiment;

[0026] Fig. 9 : Photos of rats in multiple skin irritation experiments;

[0027] Fig.10 : Photos of rats in acute toxicity experiment. DETAILED DESCRIPTION

[0028] The present invention provides a toad skin ointment for reducing swelling and relieving pain, which comprises the following components by mass: 2 to 3 parts of toad skin extract, 11 to 13 parts of vaseline, 11 to 13 parts of lanolin, 2 to 3 parts of liquid paraffin, 0.5 to 0.6 parts of glycerol, 0.3 to 0.5 parts of peppermint oil, and 0.1 to 0.3 parts of oil-soluble azone. Preferably, the components of the toad skin ointment of the present invention are 2 parts of toad skin extract, 12 parts of vaseline, 12 parts of lanolin, 3 parts of liquid paraffin, 0.5 parts of glycerol, 0.4 parts of peppermint oil, and 0.1 parts of oil-soluble azone.

[0029] The toad skin extract of the present invention is an extract of dried whole skin of Bufo bufo gargarizans Cantor, preferably an alcohol extract of dried whole skin of Bufo bufo gargarizans Cantor, preferably a freeze-dried toad skin powder equivalent to 5-6 g of toad skin original medicinal material per gram.

[0030] The preparation method of the toad skin extract of the present invention comprises the following steps: mixing the toad skin with ethanol, heating and refluxing for extraction, centrifuging the extract to obtain the supernatant, concentrating until there is no alcohol taste, and then freeze-drying to obtain toad skin freeze-dried powder. Preferably, the toad skin of the present invention is first pretreated, and the pretreatment step comprises: cutting the toad skin into pieces, placing it in a 60°C oven to dry to constant weight, and then crushing it with a pulverizer, and passing the powder through a 60-mesh sieve to obtain toad skin powder. The toad skin powder obtained by pretreatment is used for subsequent reflux extraction.

[0031] In the present invention, the solid-liquid ratio of the toad skin to ethanol is 1g:15-25mL, preferably 1g:20mL; the ethanol is a 70-90% ethanol solution, preferably an 80% ethanol solution. In the present invention, the toad skin is mixed with ethanol and then heated and refluxed for extraction.

[0032] In the present invention, the heating reflux extraction is to keep the toad skin ethanol mixture in a slightly boiling state by heating. The time of the heating reflux extraction is 2 to 3 hours, preferably 2.5 hours; the number of heating reflux extractions is 1 to 2 times. As an optional embodiment, when the number of heating reflux is 2 times, the present invention mixes the toad skin and ethanol and performs heating reflux extraction. After the first heating reflux extraction, the condensed liquid is collected and separated; ethanol is added to the toad skin raw material again and the second reflux is repeated, and the condensed liquid is collected and separated; the liquid obtained by combining the two times is the extract.

[0033] In the present invention, the centrifugal acceleration is 1700-1800×g, preferably 1760×g, the time is 15-20 min, preferably 16-18 min; the concentration temperature is 50-60°C, preferably 50-55°C; the freeze-drying temperature is -10--20°C, preferably -15°C; the freeze-drying vacuum is 150-300 Pa, preferably 200-250 Pa; the freeze-drying time is 12-24 h, preferably 15-20 h.

[0034] The toad skin extract obtained by the method of the present invention has stable contents of 5-hydroxytryptamine, bufalin, cinobufogenin and resifobufogenin, and can effectively exert the pharmacological effects of the toad skin.

[0035] The source of vaseline, lanolin, liquid paraffin, glycerol, peppermint oil, and oil-soluble azone of the present invention is not limited, and the commercially available products obtained by conventional purchase in this area can be used. In the toad skin ointment matrix of the present invention, vaseline with little skin irritation is selected, lanolin is matched, the water absorption of the ointment is improved, and drug penetration is promoted. The liquid paraffin in the ointment can adjust the viscosity of the ointment, glycerol can increase the moisture retention of the ointment, and peppermint oil and oil-soluble azone can promote the transdermal absorption of the effective ingredients in the ointment. The present invention can effectively avoid the stimulation caused by the toad skin extract to the skin by the reasonable proportion of each raw material, ensure the safety of the toad skin ointment, and promote the transdermal absorption of the effective ingredients in the toad skin extract.

[0036] The present invention also provides a method for preparing the above-mentioned toad skin ointment, comprising the following steps: vaseline, lanolin and liquid paraffin are dissolved, mixed, glycerol and toad skin extract are added, mixed evenly, peppermint oil and oil-soluble azone are added, mixed evenly, and cooled to obtain toad skin ointment. The dissolution is heated and dissolved in a water bath at 60 to 70°C, preferably heated in a water bath at 65 to 70°C. As an optional embodiment, the present invention puts vaseline, lanolin and liquid paraffin into a water bath at the same time, heats and dissolves, then slowly adds glycerol and toad skin extract, stirs and mixes evenly, then adds peppermint oil and oil-soluble azone, mixes evenly on a magnetic stirrer, and cools to obtain toad skin ointment. The toad skin ointment prepared by the method of the present invention is a slightly brownish yellow semisolid, with a particle size of <180 μm, a fine ointment, good spreadability, moderate viscosity, no sour smell, corruption, or stratification, a neutral pH, and good centrifugal, cold-resistant, and heat-resistant stability.

[0037] The present invention uses mouse ear swelling test, rat foot swelling test, mouse hot water bath tail flick test and mouse acetic acid writhing test to show that the toad skin ointment can reduce the swelling of mouse ears and rat feet, and the difference is statistically significant (P < 0.01). The percentage of increasing the pain threshold of hot water bath in mice is 71.82%, and the peripheral pain caused by acetic acid can be relieved. The writhing analgesia rate is 45.25%, indicating that the toad skin ointment has a certain inhibitory effect on inflammation, central and peripheral pain. The toad skin ointment of the present invention can significantly reduce the expression content of IL-6, TNF-α, and IL-1β in rat serum and inflammatory tissue (P < 0.01), indicating that the toad skin ointment plays an anti-inflammatory role in skin administration, and its mechanism of action may be to inhibit the NF-κB (nuclear factor κB) signaling pathway and inhibit the secretion of IL-6, TNF-α, and IL-1β. In the acetic acid writhing experiment, toad skin ointment can significantly increase (P < 0.01) the β-EP content in the serum and brain tissue of mice and reduce the PGE2 content, indicating that external application of toad skin ointment has analgesic effect, which may be achieved by inducing the synthesis of β-EP and inhibiting the secretion of PGE2.

[0038] The present invention also provides the use of the toad skin ointment or the preparation method in the preparation of anti-edema and analgesic drugs and anti-inflammatory drugs. The toad skin ointment of the present invention has a good inhibitory effect on inflammation and central and peripheral pain, and the safety experiment of the toad skin ointment shows that the toad skin ointment is non-irritating to the skin, does not cause skin edema and erythema reaction, and has high safety. The effective dosage of the toad skin ointment prepared by the present invention is 2 to 3 g / 50 kg / d, preferably 2.5 g / 50 kg / d.

[0039] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In the specific embodiment of the present invention, the experimental animals are: SPF grade KM Kunming mice, half male and half female, 4-5 weeks old, weighing (20±2) g; SPF grade SD rats, half male and half female, 6-8 weeks old, weighing (200±20) g; the experimental animals were purchased from Shandong Jinan Pengyue Experimental Animal Breeding Co., Ltd., license number: SCXK (Lu) 20220006. This animal experiment protocol was approved by the Animal Management Committee of Shandong University of Traditional Chinese Medicine, and the ethical approval number is SDUTCM20230627002.

[0041] In the specific embodiment of the present invention, the toad skin was purchased from Kangyu Chinese Medicinal Materials Co., Ltd., Rizhao City, Shandong Province, and was identified as the dried whole skin of the Chinese giant toad Bufo bufogargarizans Cantor. Glacial acetic acid (batch number: SJ00111) and xylene (batch number: FY-003) were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), prostaglandin E2 (PGE2), and β-endocrine peptide (β-EP) enzyme-linked immunosorbent assay kits were purchased from Shanghai Ke Ai Bo Biotechnology Co., Ltd., with batch numbers CB11057-Ra, CB10218-Ra, CB10205-Ra, CB10560-Mu, and CB10132-Mu, respectively. Animal tissue RNA extraction kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd., batch number: RC112-01. Erythromycin ointment was purchased from Shandong Chenxin Fodu Pharmaceutical Co., Ltd., batch number: 22103124, specification: 1%*10g / tube. Voltaren ointment was purchased from Beijing Novatis Pharmaceutical Co., Ltd., batch number: VP3037, specification: 20g:0.2g / tube. Glycerin was purchased from Jiangxi Taixinkang Industrial Co., Ltd., batch number: 20230405. Lanolin was purchased from Shandong Yousuo Chemical Technology Co., Ltd., batch number: S12741115. Vaseline was purchased from Shandong Xiekang Medical Technology Co., Ltd., batch number: 230306. Liquid paraffin was purchased from Wuyi Yanwei Medical Technology Co., Ltd., batch number: 29372513749. Bufacine (batch number: P27F10F81703), 5-hydroxytryptamine (batch number: S02HB193569), resibufogenin (C30S8G45143), and cinobucini (batch number: W27M10Z84297) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity greater than 98%.

[0042] In the specific embodiment of the present invention, the instruments used are PE28 pH meter (Mettler-Toledo Instrument Co., Ltd.), Agilent 1260 high performance liquid chromatograph (1260 Infinity II diode array detector HS, 1260 Infinity II high capacity column oven, Agilent); Alpha 1-4LDplus freeze dryer (Shanghai Shuangxu Electronics Co., Ltd.), ST8R high-speed refrigerated centrifuge (Thermo Fisher Scientific Co., Ltd.), HH-S6 water bath (Changzhou Guoyu Instrument Manufacturing Co., Ltd.), 79-1 magnetic heating stirrer (Jintan Zhongda Instrument Factory), PV-200 toe volume meter (Chengdu Taimeng Software Co., Ltd.), CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad Life Sciences Products Co., Ltd.).

[0043] In a specific embodiment of the present invention, the matrix used in the matrix group is composed of 12g of vaseline, 12g of lanolin, 3g of liquid paraffin, 0.5g of glycerol, 0.4g of peppermint oil, and 0.1g of oil-soluble azone. The preparation method is as follows: vaseline, lanolin, and liquid paraffin are dissolved in a 70°C water bath, glycerol is slowly added, stirred evenly, peppermint oil and oil-soluble azone are added, and mixed evenly on a magnetic stirrer and then cooled to make.

[0044] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0045] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0046] Example 1

[0047] 1. Research on the dosage of vaseline, lanolin, glycerin and liquid paraffin

[0048] Preparation of toad skin extract: Cut the toad skin into pieces, place it in a 60℃ oven and dry it to constant weight, then crush it with a grinder, and pass the powder through a 60-mesh sieve to obtain toad skin powder. Toad skin powder and ethanol are added to 80% ethanol solution according to the material-liquid ratio of 1g:20mL, and heated reflux extraction is performed twice, each time for 2.5h: after the first heating reflux extraction, the condensed liquid is collected and separated; the toad skin raw material is added with an equal amount of 80% ethanol solution again and refluxed for the second time, and the condensed liquid is collected and separated; the liquid obtained by combining the two times is the extract. The extract is centrifuged at 1760×g for 15min and the supernatant is taken, concentrated at 50℃ until there is no alcohol taste, and then freeze-dried (temperature is -15℃, vacuum degree is 200Pa, time is 20h). The toad skin freeze-dried powder equivalent to 5g of the original toad skin medicinal material per gram is obtained and stored at 4℃ for use.

[0049] Preparation method of toad skin ointment: dissolve vaseline, lanolin and liquid paraffin in a 70°C water bath, slowly add glycerin and 2 g of toad skin extract, stir evenly, add 0.4 g of peppermint oil and 0.1 g of oil-soluble azone, mix evenly on a magnetic stirrer and let cool to make toad skin ointment.

[0050] The dosage of vaseline (A), lanolin (B), glycerin (C) and liquid paraffin (D) was selected as the influencing factors, and the sensory evaluation (a), centrifugal stability (b), heat stability (c) and cold stability (d) of the ointment were used as evaluation indicators for scoring. The sensory evaluation method is as follows: take each group of toad skin ointment and check its color, spreadability, fineness and stratification. The stability inspection method is as follows: take 3 portions of toad skin ointment from each group, place at -20℃ for 24h (cold stability), place at 45℃ for 24h (heat stability), and centrifuge at 990×g for 20min (centrifugal stability). After the experiment, the cold stability, heat stability and centrifugal stability of the toad skin ointment were checked by observing whether the toad skin ointment was stratified, whether the appearance of the ointment changed, and whether the drug was precipitated. The scoring rules are shown in Table 1, and the levels of each factor in the uniform design and the scoring results are shown in Table 2.

[0051] Table 1 Ointment scoring rules

[0052]

[0053] Table 2 Uniform design experimental results

[0054]

[0055]

[0056] IBM SPSS Statistics26 was used to perform regression analysis on the experimental results. The linear regression results are shown in Tables 3 and 4.

[0057] Table 3 Analysis of variance

[0058] project Degrees of Freedom Sum of Squares SS Mean Square F Significance Regression analysis 4 2108.054 527.014 19.174 0.007 Residual 4 109.946 27.486 total 8 2218.000

[0059] Table 4 Parameters

[0060] project coefficient Standard error T-test P-value Lower limit 95.0% Upper limit 95.0% intercept 56.947 10.002 5.694 0.0047 29.178 84.717 Vaseline 1.876 0.501 3.743 0.0200 0.484 3.267 Lanolin 2.208 0.400 5.516 0.0053 1.096 3.319 Liquid paraffin -1.155 0.805 -1.434 0.2249 -3.390 1.081 glycerin -13.487 2.108 -6.399 0.0031 -19.339 -7.635

[0061] The regression equation is Y = 56.947 + 1.876X1 + 2.208X2 - 1.155X3 - 13.487X4, and the linear correlation coefficient is R = 0.975, R 2=0.950, the overall significance test value F=19.174, the significance level P=0.007<0.01, indicating that the linearity is credible and the equation is significant. According to the linear regression equation, the coefficients of vaseline and lanolin are positive, indicating that the comprehensive score Y value increases with the increase, and the coefficients of liquid paraffin and glycerol are negative, indicating that the comprehensive score Y value decreases with the decrease. And the influence of each factor on the comprehensive score is glycerol (D)> lanolin (B)> vaseline (A)> liquid paraffin (C). Therefore, in order to obtain a higher comprehensive score, the amount of vaseline and lanolin needs to be larger, and the amount of liquid paraffin and glycerol needs to be smaller. According to the results and after comprehensive consideration of the later design and experimental verification, the optimal matrix prescription composition of toad skin ointment is: 12g vaseline, 12g lanolin, 3g liquid paraffin, and 0.5g glycerol per 30g toad skin ointment. According to the linear equation, the comprehensive score under this condition is 95.75.

[0062] 2. Verification experiment:

[0063] The prescription of toad skin ointment is: 2g toad skin extract, 12g vaseline, 12g lanolin, 3g liquid paraffin, 0.5g glycerin, 0.4g peppermint oil, 0.1g oil-soluble azone. Preparation method of toad skin ointment: dissolve vaseline, lanolin, and liquid paraffin in a 70°C water bath, slowly add glycerin and toad skin extract, stir evenly, add peppermint oil and oil-soluble azone, mix evenly on a magnetic stirrer, and cool to make toad skin ointment.

[0064] Three batches of toad skin ointment were prepared respectively, and the sensory, centrifugal stability, cold stability, and heat stability were evaluated according to the method described in step 1. The comprehensive score was calculated, and the final comprehensive score was (95.67±0.58) points, which was close to the expected value of 95.75 points of the equation, indicating that the model had a good fit and the prescription was reasonable.

[0065] 3. Quality evaluation of toad skin ointment

[0066] Quality inspection of 3 batches of toad skin ointment prepared in step 2

[0067] (1) Property evaluation: Three batches of toad skin ointment were taken and their color, spreadability, smoothness, and stratification were examined.

[0068] The results showed that the toad skin ointment was a slightly brownish-yellow semisolid with fine texture, good spreadability, moderate viscosity, and no sour smell, corruption, or stratification.

[0069] (2) Determination of pH value of ointment: Take appropriate amount of toad skin ointment from three batches, add distilled water in a ratio of 1 g:10 mL in a beaker, heat and stir continuously until melted, filter after cooling, and measure with a pH meter. Each group is measured three times in parallel, and the average value is the pH value of the toad skin ointment.

[0070] The pH values ​​of the three batches of toad skin ointment were 7.02, 7.11, and 7.23, respectively, and the RSD value among the three batches of toad skin ointment was 1.48%.

[0071] (3) Particle size: Take an appropriate amount of toad skin ointment from three batches, place it on a glass slide and cover it with a cover glass, press gently to make the ointment particles evenly distributed, and observe under a microscope.

[0072] Under the microscope, the particle size of the three batches of toad skin ointment was an average of <180μm, which meets the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0073] (4) Determination of viscosity: Take 20 g of three batches of toad skin ointment and put them in a clean small beaker. Use a viscometer to measure the viscosity. The measurements were repeated three times and the average value was taken as the viscosity of the toad skin ointment.

[0074] The average viscosities of the three batches of ointment were 5094.67 MPa.s, 5080 MPa.s, and 5072.67 MPa.s, respectively. The RSD values ​​of each batch of toad skin ointment were 0.68%, 0.68%, and 0.36%, respectively.

[0075] (5) The stability test method is as follows: 3 portions of 3 batches of toad skin ointment are taken, placed at -20°C for 24 h, 45°C for 24 h, and centrifuged at 990×g for 20 min. After the experiment, the cold stability, heat stability, and centrifugal stability of the toad skin ointment are tested by observing whether the toad skin ointment is stratified, whether the appearance of the ointment changes, and whether the drug precipitates.

[0076] In the cold-resistance, heat-resistance and centrifugation test results of three batches of toad skin ointment, no stratification or drug precipitation occurred in the ointment. After the experiment, the ointment had uniform color, good spreadability and fine texture, indicating that the toad skin ointment had good centrifugation, cold-resistance and heat-resistance stability.

[0077] 4. Determination of the content of toad skin ointment

[0078] The toad skin ointment prepared in step 2 was assayed

[0079] (1) Chromatographic conditions:

[0080] Bufon venom lactone components: Agilent InfinityLab Poroshell 120EC-C18 (150mm×3mm 2.7μm) chromatographic column; mobile phase A was 0.1% phosphoric acid water, mobile phase B was acetonitrile, gradient elution (0-5min, 80%→75%A; 5-20min, 75%→71%A; 20-35min, 71%→65%A; 35-50min, 65%→60%A), column temperature 30°C, detection wavelength 296nm, flow rate 0.5mL / min, injection volume 5μL, automatic injection.

[0081] 5-HT: Diamonsil Plus C18 (250 mm × 4.6 mm, 5 μm) chromatographic column; the mobile phase was 0.05 mol / L potassium dihydrogen phosphate-acetonitrile (96:4, pH = 6.83) solution, isocratic elution, column temperature was 30 ° C, flow rate was 0.8 mL / min, detection wavelength was 275 nm, injection was 5 μL, and automatic injection was used.

[0082] (2) Preparation of test solution: Accurately weigh 5 g of toad skin ointment, add 50 mL of 80% ethanol, reflux and extract for 1 h, cool, make up for weight loss, centrifuge at 1760 × g for 15 min, remove the supernatant, filter through a membrane, and obtain the filtrate.

[0083] (3) Preparation of reference solution: Accurately weigh 5-HT, cinobufogenin, resifolinin, and bufalin standards, dissolve them in anhydrous ethanol to a concentration of 1 mg / mL, and shake well.

[0084] (4) Preparation of negative sample solution: Take blank matrix, add anhydrous ethanol to dissolve it to a mass concentration of 1 mg / mL, and shake well.

[0085] Linear relationship investigation: Take the solution under (3), add anhydrous ethanol to prepare different concentrations, and draw a standard curve. The standard curves of bufalin, cinobufogenin, resifugine and 5-hydroxytryptamine are: Y1=29.991X1+97.014, Y2=9.0219X2-22.927, Y3=7.7877X3-4.723, Y4=6.552X4-4.4513,

[0086] Precision investigation: Take the test solution under item (2) and make six injections under the conditions of item (1). The average contents of bufalotoxin, cinobuccal venom toxin base, resifobacillus genistein and 5-hydroxytryptamine are 0.023, 0.048, 0.045 and 0.032 mg / g, respectively. The RSD values ​​of the six measurements are 1.89%, 0.94%, 1.61% and 1.04%, respectively, indicating that the precision of the instrument is good.

[0087] Stability study: Take the test sample solution under item (2) and inject it for determination 6 times at 0, 2, 4, 8, 12 and 24 h according to the method of item (1). The RSDs of the peak areas of bufalotoxin, cinobuccal venom toxin base, resifobacillus genistein and 5-hydroxytryptamine are 1.67%, 1.00%, 1.54% and 1.12%, respectively, indicating good stability.

[0088] Repeatability study: Weigh 6 portions of toad skin ointment accurately and prepare the test solution according to the method in item (2). Samples were injected and measured 6 times according to the method in item (1). The average contents of bufalin, cinobucoidin, resifobacillus genin and 5-hydroxytryptamine were 0.023, 0.048, 0.044 and 0.031 mg / g, and the RSDs of the contents were 1.65%, 1.83%, 2.33% and 1.18%, respectively.

[0089] Sample addition recovery rate: Take 6 portions of toad skin ointment with known content (5 g), accurately add appropriate amount of toad venom, cinobucoid venom base, resibovagin and 5-hydroxytryptamine reference solution, prepare the test solution according to method (2), and inject and measure 6 times according to method (1), determine the content and calculate the recovery rate. The average recoveries of toad venom, cinobucoid venom base, resibovagin and 5-hydroxytryptamine are 95.36%, 99.02%, 97.62% and 104.99%, and their RSDs are 1.07%, 1.41%, 2.40% and 2.28%, respectively.

[0090] Specificity: Take the negative sample solution, and measure the bufotoxin, cinobufolin, resifobafugin, and 5-HT reference solution according to the chromatographic method in item (1). The chromatogram is as follows: Figure 1 As shown. In the figure, A, B, and C represent the chromatograms of negative sample solution (determination of toad venom lactone components), toad skin ointment sample solution (determination of toad venom lactone components), and reference solution (buflin, cinobufolin, and resifobafoxin) respectively; a, b, and c are chromatograms of negative sample solution (determination of 5-hydroxytryptamine content), toad skin ointment sample solution (determination of 5-hydroxytryptamine content), and chromatograms of 5-hydroxytryptamine reference solution; 1, 2, 3, and 4 represent buflin, resifobafoxin, cinobufolin, and 5-hydroxytryptamine respectively. This indicates that the negative sample solution has no interference with the determination of the index component.

[0091] Three batches of toad skin ointment were taken for content determination. The results of the content determination of 5-hydroxytryptamine, bufalin, cinobufolin and resifobafugin in the ointment are shown in Table 5.

[0092] Table 5 Content determination results

[0093] name 1 batch content (mg / g) 2 batches of content (mg / g) 3 batches of content (mg / g) Average value (mg / g) RSD value (%) Bufalin 0.0230 0.0224 0.0228 0.0227 1.29 Cinobufacini 0.0495 0.0485 0.0483 0.0488 1.27 Fattoad 0.0460 0.0447 0.0456 0.0454 1.47 Serotonin 0.0318 0.0317 0.0321 0.0318 0.701

[0094] Example 2

[0095] Preparation of toad skin extract: Cut the toad skin into pieces, place it in a 60℃ oven and dry it to constant weight, then crush it with a grinder, and pass the powder through a 60-mesh sieve to obtain toad skin powder. Toad skin powder and ethanol are added to 80% ethanol solution according to the material-liquid ratio of 1g:20mL, and heated reflux extraction is performed twice, each time for 2.5h: after the first heating reflux extraction, the condensed liquid is collected and separated; the toad skin raw material is added with an equal amount of 80% ethanol solution again and refluxed for the second time, and the condensed liquid is collected and separated; the liquid obtained by combining the two times is the extract. The extract is centrifuged at 1760×g for 15min and the supernatant is taken, concentrated at 50℃ until there is no alcohol taste, and then freeze-dried (temperature is -15℃, vacuum degree is 200Pa, time is 20h). The toad skin freeze-dried powder equivalent to 5g of the original toad skin medicinal material per gram is obtained and stored at 4℃ for use.

[0096] The prescription of toad skin ointment is: 2g toad skin extract, 12g vaseline, 12g lanolin, 3g liquid paraffin, 0.5g glycerin, 0.4g peppermint oil, 0.1g oil-soluble azone. Preparation method of toad skin ointment: dissolve vaseline, lanolin, and liquid paraffin in a 70°C water bath, slowly add glycerin and toad skin extract, stir evenly, add peppermint oil and oil-soluble azone, mix evenly on a magnetic stirrer, and cool to make toad skin ointment.

[0097] Example 3

[0098] Preparation of toad skin extract: Cut the toad skin into pieces, place it in a 60℃ oven to dry to constant weight, then crush it with a grinder, and pass the powder through a 60-mesh sieve to obtain toad skin powder. Toad skin powder and ethanol are added according to a solid-liquid ratio of 1g:15mL, 70% ethanol solution is added, and heated reflux extraction is performed once for 2.5h. The condensed liquid is collected and separated to obtain the extract. The extract is centrifuged at 1700×g for 20min, the supernatant is taken, concentrated at 60℃ until there is no alcohol taste, and then freeze-dried (temperature is -15℃, vacuum degree is 200Pa, time is 20h). The toad skin freeze-dried powder equivalent to 5.2g of toad skin original medicinal material per gram is obtained, and stored at 4℃ for later use.

[0099] The prescription of toad skin ointment is: 2g toad skin extract, 11g vaseline, 11g lanolin, 2g liquid paraffin, 0.5g glycerin, 0.3g peppermint oil, 0.1g oil-soluble azone. Preparation method of toad skin ointment: dissolve vaseline, lanolin, and liquid paraffin in a 60°C water bath, slowly add glycerin and toad skin extract, stir evenly, add peppermint oil and oil-soluble azone, mix evenly on a magnetic stirrer, and cool to make toad skin ointment.

[0100] Example 4

[0101] Preparation of toad skin extract: Cut the toad skin into pieces, place it in a 60℃ oven to dry to constant weight, then crush it with a grinder, and pass the powder through a 60-mesh sieve to obtain toad skin powder. Toad skin powder and ethanol are added to a 90% ethanol solution at a solid-liquid ratio of 1g:25mL, and heated reflux extraction is performed once for 3h. The condensed liquid is collected and separated to obtain the extract. The extract is centrifuged at 1800×g for 15min, the supernatant is taken, concentrated at 55℃ until there is no alcohol taste, and then freeze-dried (temperature is -15℃, vacuum degree is 200Pa, time is 20h). The toad skin freeze-dried powder equivalent to 5.6g of the original toad skin medicinal material per gram is obtained, and stored at 4℃ for later use.

[0102] The prescription of toad skin ointment is: 3g toad skin extract, 13g vaseline, 13g lanolin, 3g liquid paraffin, 0.6g glycerin, 0.5g peppermint oil, 0.3g oil-soluble azone. Preparation method of toad skin ointment: dissolve vaseline, lanolin, and liquid paraffin in a 65°C water bath, slowly add glycerin and toad skin extract, stir evenly, add peppermint oil and oil-soluble azone, mix evenly on a magnetic stirrer, and cool to make toad skin ointment.

[0103] Example 5

[0104] Preparation of toad skin extract: Cut the toad skin into pieces, place it in a 60℃ oven to dry to constant weight, then crush it with a grinder, and pass the powder through a 60-mesh sieve to obtain toad skin powder. Toad skin powder and ethanol are added to a 90% ethanol solution according to a solid-liquid ratio of 1g:15mL, and heated reflux extraction is performed twice, each time for 2h: after the first heating reflux extraction, the condensed liquid is collected and separated; the toad skin raw material is added with an equal amount of 90% ethanol solution again and refluxed for the second time, and the condensed liquid is collected and separated; the liquid obtained by combining the two times is the extract. The extract is centrifuged at 1700×g for 20min, the supernatant is taken, concentrated at 55℃ until there is no alcohol taste, and then freeze-dried (temperature is -15℃, vacuum degree is 200Pa, time is 20h). The toad skin freeze-dried powder equivalent to 5.8g of the original toad skin medicinal material per gram is obtained, and stored at 4℃ for use.

[0105] The prescription of toad skin ointment is: 2g toad skin extract, 12g vaseline, 12g lanolin, 2g liquid paraffin, 0.6g glycerin, 0.4g peppermint oil, 0.2g oil-soluble azone. Preparation method of toad skin ointment: dissolve vaseline, lanolin, and liquid paraffin in a 60°C water bath, slowly add glycerin and toad skin extract, stir evenly, add peppermint oil and oil-soluble azone, mix evenly on a magnetic stirrer, and cool to make toad skin ointment.

[0106] Example 6

[0107] Preparation of toad skin extract: Cut the toad skin into pieces, place it in a 60℃ oven to dry to constant weight, then crush it with a grinder, and pass the powder through a 60-mesh sieve to obtain toad skin powder. Toad skin powder and ethanol are added to 75% ethanol solution according to the material-liquid ratio of 1g:25mL, and heated reflux extraction is performed twice, each time for 3h: after the first heating reflux extraction, the condensed liquid is collected and separated; the toad skin raw material is added with an equal amount of 75% ethanol solution again and refluxed for the second time, and the condensed liquid is collected and separated; the liquid obtained by combining the two times is the extract. The extract is centrifuged at 1800×g for 15min, the supernatant is taken, concentrated at 60℃ until there is no alcohol taste, and then freeze-dried (temperature is -15℃, vacuum degree is 200Pa, time is 20h). The toad skin freeze-dried powder equivalent to 5.4g of toad skin raw material per gram is obtained, and stored at 4℃ for use.

[0108] The prescription of toad skin ointment is: 3g toad skin extract, 11g vaseline, 13g lanolin, 2.5g liquid paraffin, 0.6g glycerin, 0.3g peppermint oil, 0.2g oil-soluble azone. Preparation method of toad skin ointment: dissolve vaseline, lanolin, and liquid paraffin in a 70°C water bath, slowly add glycerin and toad skin extract, stir evenly, add peppermint oil and oil-soluble azone, mix evenly on a magnetic stirrer, and cool to make toad skin ointment.

[0109] Test Example 1

[0110] Evaluation of the detumescence efficacy of the toad skin ointment prepared in Example 2

[0111] 1. Xylene-induced ear swelling experiment in mice

[0112] 40 healthy mice were randomly divided into 4 groups, namely model control group, matrix group, toad skin ointment group, and erythromycin ointment group (positive drug group), with 10 mice in each group, half male and half female. The model control group was given normal saline, and the matrix group, toad skin ointment group, and erythromycin ointment group were given blank matrix, toad skin ointment, and erythromycin ointment, respectively, which were applied to both sides of the right ear of each mouse, 0.1g / mouse, twice a day, for 7 consecutive days. One hour after the last application of the drug, 200μL of xylene was applied to the right ear of each mouse to cause inflammation. During the entire experiment, the left ear of the mouse was not treated in any way and served as the normal control ear. One hour later, the mouse was anesthetized and killed by dislocation of the neck, and both ears were cut off. A mouse puncher with a diameter of 6mm was used to punch holes in the same part of the left and right ears of the mouse, and its weight was accurately weighed.

[0113] Mouse ear swelling = right ear piece weight (coated with xylene) - left ear piece weight (control)

[0114] Ear swelling rate (%) = (mouse ear swelling / left ear piece weight) × 100%

[0115] The results are as follows Figure 2As shown in the figure, compared with the model control group, **P<0.01; *P<0.05. Figure 2 It can be seen that the differences in ear swelling and ear swelling inhibition rate between the toad skin ointment group, the positive drug group and the model control group were statistically significant (P<0.01), and the ear swelling degree between the matrix group and the model control group was not statistically significant (P>0.05). Both the swelling degree and ear swelling inhibition rate experimental results showed that the toad skin ointment group had the best detumescence effect, which was better than the positive drug group.

[0116] 2. Rat carrageenan foot swelling experiment

[0117] Twenty-four healthy rats were randomly divided into four groups: model control group, matrix group, erythromycin ointment group (positive drug group), and toad skin ointment group, with 6 rats in each group, half of them were male and half were female. The model control group was given normal saline, and the matrix group, toad skin ointment group, and erythromycin ointment group were given blank matrix, toad skin ointment, and erythromycin ointment, respectively. The right hind paw of the rats in each group was given 0.1 g / rat, twice a day, for 7 consecutive days. One hour after the last application of the drug, the right hind paw volume of the rats was measured after anesthesia. Then, 1% carrageenan 0.1 mL / rat was subcutaneously injected into the right hind paw to induce inflammation. The paw volume was measured with a toe volume meter at 0.5 h, 1.5 h, and 3 h after inflammation, and the swelling degree and inhibition rate were calculated. The differences in swelling degree among the groups were compared.

[0118] Swelling degree (mL) = volume of the foot after inflammation (mL) - volume of the foot before inflammation (mL)

[0119] Swelling inhibition rate (%) = (average swelling degree of model control group - average swelling degree of drug administration group) / average swelling degree of model control group × 100%

[0120] The results are as follows Figure 3 As shown in the figure, compared with the model control group, **P < 0.01; *P < 0.05, a, b, c represent 0.5h, 1h, 3h after inflammation, respectively. Figure 3 It can be seen that there was no significant difference in swelling between the matrix group and the model control group (P>0.05). Compared with the model control group, the swelling of the rat feet in the toad skin ointment group and the positive drug group was significantly reduced, and the difference was statistically significant (P<0.01). It proves that the toad skin ointment group has a significant inhibitory effect on rat foot swelling.

[0121] 3. Detection of TNF-α, IL-6, and IL-1β protein levels in serum and tissues by enzyme-linked immunofluorescence technique

[0122] After the rat foot swelling experiment in step 2 was completed, rats that were not treated were used as the blank control group. About 1 mL of blood was collected from the eye sockets of the rats in the model control group, positive drug group, and toad skin ointment group. After standing at 4°C overnight, the blood was centrifuged at 1348×g for 15 minutes, and the supernatant was stored in a -80°C refrigerator. The swollen foot tissues of rats in the model control group, positive drug group, and toad skin ointment group were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for the determination of TNF-α, IL-6, and IL-1β protein content. The supernatant was taken after homogenization for later use. According to the instructions of the enzyme-linked immunosorbent assay (ELISA) test kit, the TNF-α, IL-6, and IL-1β protein content in serum and inflammatory tissues was detected.

[0123] The results are as follows Figure 4 As shown in the figure, the blank control group was compared with the model control group, ##P<0.01, #P<0.05; the experimental group was compared with the model control group, **P<0.01; *P<0.05. Figure 4 It can be seen that compared with the blank control group, the protein levels of TNF-α, IL-6, and IL-1β in the serum and inflammatory tissues of the model control group were significantly increased, and the difference was statistically significant (P<0.01), indicating that carrageenan can cause inflammatory response in rats. Compared with the model control group, the positive drug group, the toad skin ointment group, can significantly reduce (P<0.01) the levels of TNF-α, IL-6, and IL-1β in the serum and inflammatory tissues of rats with edema of the foot. The above results indicate that the external use of toad skin ointment may reduce the inflammatory response by reducing the secretion levels of TNF-α, IL-6, and IL-1β.

[0124] 4. qRT-PCR detection of the expression of TNF-α, IL-6, and IL-1β in rat inflammatory tissues in the foot swelling experiment

[0125] The swollen foot tissues of the model control group, positive drug group, and toad skin ointment group after the rat foot swelling experiment in step 2 were taken, and the rats without any treatment were used as the blank control group. After grinding with liquid nitrogen, total RNA was extracted according to the animal tissue RNA extraction kit, and the TB Green Premix Ex TaqTMⅡ(Tli RNaseH Plus) kit was used for reversal, and finally the PrimeScripTM RT reagent Kit with gDNA Eraser(Perfect Real Time) kit was used for qRT-PCR detection. The primer sequences are shown in Table 6.

[0126] Table 6 Primer sequences

[0127]

[0128]

[0129] The results are as follows Figure 5 As shown in the figure, the blank control group was compared with the model control group, ##P<0.01, #P<0.05; the experimental group was compared with the model control group, **P<0.01; *P<0.05. Figure 5 It can be seen that in the detection of three inflammatory factors, the expression levels of the three inflammatory factors in the positive drug and toad skin ointment groups were statistically significant different from those in the model control group (P<0.01, P<0.05). This further verified that the anti-inflammatory effect of toad skin ointment may be related to reducing the expression levels of TNF-α, IL-6, and IL-1β in the body.

[0130] Test Example 2

[0131] Study and evaluation of the analgesic efficacy of the toad skin ointment prepared in Example 2

[0132] 1. Mouse hot water bath tail flick experiment

[0133] 40 healthy mice were randomly divided into 4 groups, namely, model control group, matrix group, positive drug group (Voltaren ointment group), and toad skin ointment group, with 10 mice in each group, half male and half female. The model control group was given normal saline, and the matrix group, toad skin ointment group, and Voltaren ointment group were given blank matrix, toad skin ointment, and Voltaren ointment, respectively. Each group was given 0.1g / mouse, twice a day, for 7 consecutive days, and 1 hour after the last administration, the mouse tail was immersed in 50℃ water to measure its pain threshold (the time from the tail entering the water to being thrown out). The results are shown in Table 7.

[0134] Pain threshold increase percentage (%) = (pain response time of drug administration group (s) - pain response time of model control group (s)) / pain response time of model control group (s) × 100%

[0135] Table 7 Effect of toad skin ointment on the tail flick threshold of mice in hot water bath

[0136] Grouping Threshold(s) Pain threshold increase percentage (%) Model control group 3.14±0.61 —— Matrix Group 3.50±0.635 11.68 Toad skin ointment group 5.32±0.67** 71.82 Positive drug group 5.68±0.88** 82.55

[0137] As shown in Table 7, compared with the model control group, the positive drug group and the toad skin ointment group can significantly increase the tail-flick threshold of mice, and the difference is statistically significant (P<0.01). The percentages of toad skin ointment and positive drug on the hot water bath tail-flick pain threshold of mice are 71.82% and 82.55% respectively, which proves that toad skin ointment has a good analgesic effect.

[0138] 2. Acetic acid-induced acute pain in mice writhing experiment

[0139] 50 healthy mice were randomly divided into 5 groups, namely blank control group, model control group, matrix group, Voltaren ointment group, and toad skin ointment group, with 10 mice in each group, half male and half female. The blank control group was not treated in any way, the model control group was given normal saline, the matrix group, toad skin ointment group, and Voltaren ointment group were given blank matrix, toad skin ointment, and Voltaren ointment respectively, and each group was administered at the tail of each mouse, 0.1g / mouse, 2 times / day, for 7 consecutive days, and 1h after the last administration, each group was intraperitoneally injected with 200μL of 0.5% glacial acetic acid solution. The time of the first occurrence of the writhing phenomenon (abdominal twitching, the tail root rising is a complete writhing reaction) of each group of mice and the number of writhing times of each group of mice in 15min were observed and the analgesic rate of each group of mice was calculated. The results are shown in Table 8.

[0140] Mouse analgesia rate (%) = (number of writhings in the model control group - number of writhings in the drug-treated group) / number of writhings in the model control group × 100%

[0141] Table 8 Effect of toad skin ointment on acetic acid writhing in mice

[0142] Grouping First twist time (s) Number of twists (times) Analgesia rate (%) Blank control group —— —— —— Model control group 180.90±31.75 63.20±8.18 —— Matrix Group 185.28±10.48 52.60±13.22 9.49 Toad skin ointment group 228.53±23.78 34.60±4.53** 45.25 Positive drug group 255.48±87.91* 29.30±6.17** 53.64

[0143] As shown in Table 8, the time of the first writhing of mice was statistically significant between the positive drug group and the model control group (P < 0.05), and there was no significant difference between the toad skin ointment group and the model control group (P> 0.05), but the toad skin ointment and the positive drug significantly reduced (P < 0.01) the number of writhing times of mice within 15 minutes, and the analgesic rates were 45.25% and 53.64%, respectively. The blank control was not treated in any way, so there was no writhing and no data.

[0144] 3. Detection of β-EP and PGE2 protein content in serum and tissues by enzyme-linked immunofluorescence technique

[0145] After the acetic acid writhing experiment in step 2, the mice in the model control group, positive drug group, and toad skin ointment group were anesthetized, and the eyeballs were removed to collect about 0.5 mL of blood. The blood was centrifuged at 1348×g for 15 min and the supernatant was collected for later use. The brain tissue homogenate was centrifuged and the supernatant was collected for later use. The β-EP and PGE2 protein contents in the serum and brain tissue supernatant were detected according to the instructions of the ELISA detection kit. The mice without any treatment were used as the blank control group.

[0146] The results are as follows Figure 6As shown in the figure, the blank control group was compared with the model control group, ##P<0.01, #P<0.05; the experimental group was compared with the model control group, **P<0.01; *P<0.05. Compared with the blank group, the β-EP in the serum and cerebral cortex of the model control group was significantly reduced, and PGE2 was significantly increased, and the difference was statistically significant (P<0.01); compared with the model control group, the β-EP content in the cerebral cortex and serum of the mice in the toad skin ointment group and the positive drug group was significantly increased, and the difference was statistically significant (P<0.01), and the PGE2 content was significantly decreased, with a very significant difference (P<0.01).

[0147] 4. qRT-PCR detection of POMC and PGE2 gene expression in brain tissue

[0148] After the acetic acid writhing experiment in step 2, the model control group, positive drug group, and toad skin ointment group were used to detect the mRNA expression levels of PGE2 and β-EP precursor pro-opiomelanocortin (POMC) in the brain tissue of the pain model mice by mouse qRT-PCR: the brain tissue of each group of mice stored in a -80°C refrigerator was taken, and the total RNA was extracted according to the animal tissue RNA extraction kit after liquid nitrogen grinding, and the TB Green Premix Ex TaqTMⅡ(Tli RNaseH Plus) kit was used for reversal, and finally the PrimeScripTM RT reagent Kit with gDNA Eraser(Perfect Real Time) kit was used for qRT-PCR detection. The mice without any treatment were used as the blank control group. The primer sequences are shown in Table 9.

[0149] Table 9 Primer sequences

[0150]

[0151] The results are as follows Figure 7 As shown in the figure, the blank control group was compared with the model control group, ##P<0.01, #P<0.05; the experimental group was compared with the model control group, **P<0.01; *P<0.05. Compared with the model control group, the positive drug group and the toad skin ointment group significantly increased the mRNA expression level of POMC in the brain tissue of mice (P<0.05, P<0.01). In terms of the difference in the expression level of PGE2 mRNA in the brain tissue of mice in each group, compared with the model control group, the toad skin ointment and the positive drug can significantly reduce the mRNA expression level of PGE2, and the difference is statistically significant (P<0.01).

[0152] Test Example 3

[0153] Experimental study on the safety of the toad skin ointment prepared in Example 2

[0154] 1. Single-dose skin irritation test

[0155] 24 healthy rats were divided into 4 groups (intact skin toad skin ointment group, intact skin blank matrix group, damaged skin toad skin ointment group and damaged skin blank matrix group), 6 rats in each group, half male and half female. Before administration, the back of each group of rats was depilated. The intact skin experimental group was careful not to scratch the skin. The left side of the back skin of the rats in the damaged skin experimental group was scratched with a syringe needle in the shape of "#". When applying the medicine, 0.1g of toad skin ointment and blank matrix were taken and applied to the left side of the back skin of the rats respectively. The right side of the back of the rats was the blank control area, and an equal amount of normal saline was applied. After 24h of administration, the residual ointment was removed with warm water. The situation of the application site on the back of the rats was observed at 1, 24, 48 and 72h, and the skin irritation reaction was scored according to Table 10. The skin irritation intensity of rats was evaluated according to Table 11. The highest score of erythema edema was 8 points.

[0156] Table 10 Scoring criteria for skin irritation reaction

[0157] erythema Edema Points No erythema No edema 0 Barely visible erythema Barely visible edema 1 Visible erythema Visible edema 2 Moderate to severe erythema The skin is raised 1mm with clear contours 3 Purple-red erythema with eschar formation Edema >1mm 4

[0158] Table 11 Skin irritation intensity evaluation table

[0159]

[0160]

[0161] In the single-dose skin irritation experiment, the skin irritation score of rats in each group was 0. Figure 8 As shown in the figure, A and B represent the intact skin toad skin ointment group and the blank matrix group, respectively; a and b represent the damaged skin toad skin ointment group and the blank matrix group, respectively. The left side of the rat's back is the experimental area, and the right side is the blank control area. The results of the single-dose skin irritation experiment showed that there was no difference between the drug application site on the back of the rats in the intact skin group and the control area on the right side, with no erythema or swelling reaction. The "#" area on the back of the rats in the damaged skin group was scabby, and no edema or erythema was observed in the other parts.

[0162] 2. Repeated administration skin irritation test

[0163] The experimental grouping and application method were the same as the single-dose skin irritation experimental method in step 1. The drug was applied to the same part of the rat's back for 7 consecutive days, once a day. The skin irritation reaction of the rat's back was observed at the same time every day and scored. The skin irritation reaction score was performed according to Table 10, and the rat skin irritation intensity was evaluated according to Table 11. The maximum total score of erythema and edema was 8 points.

[0164] In the multiple-dose skin irritation experiment, the skin irritation score of rats in each group was 0. Fig. 9As shown, there was no skin irritation in the damaged skin toad skin ointment group and the intact skin toad skin ointment group during the experiment (1 to 7 days), and the back skin of the rats in each experimental group was smooth, without erythema or edema. In the damaged skin group, scabs formed on the first day, and the scabs began to fall off on the fourth day, and the scabs were basically completely shed on the seventh day.

[0165] 3. Acute skin toxicity test

[0166] Thirty-six healthy SD rats were divided into six groups (toad skin ointment intact skin experimental group and damaged skin experimental group; blank matrix intact skin experimental group and damaged skin experimental group; blank control group intact skin experimental group and damaged skin experimental group), with 6 rats in each group, half male and half female. The back of the rats was depilated one day before the experiment. The rats with intact skin without damage after depilation were used as the intact skin experimental group. After the depilation of the damaged skin experimental group, the middle area of ​​the back of the rats was scratched in the shape of a "#". When applying the medicine, 0.1g of toad skin ointment and blank matrix without toad skin extract were applied to the depilated area on the back of the rats. The blank control group applied physiological saline to the same area once a day for 7 consecutive days. The rats in the experimental group were observed every day for systemic poisoning and death, and the diet and weight gain of each group were recorded.

[0167] During the acute toxicity experiment, the mice's hair, feces, urine, diet, drinking water, and activities were normal, and no poisoning or death occurred. The back skin of the rats in the intact skin group was smooth, without redness or swelling. The damaged area on the back of the rats in the damaged skin group went from scabs to scabs that began to fall off and then to scabs that fell off completely, without erythema or swelling. Fig.10 As shown in Table 12, the weight gain of rats in each group was not significantly different from that of the blank control group (P>0.05). No organ lesions were found at the end of the experiment, indicating that the toad skin ointment has low toxicity and good safety for skin medication.

[0168] Table 12 Rat body weight weighing results

[0169]

[0170]

[0171] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A toad skin ointment for reducing swelling and relieving pain, characterized in that: The composition comprises the following components by weight: 2-3 parts of toad skin extract, 11-13 parts of vaseline, 11-13 parts of lanolin, 2-3 parts of liquid paraffin, 0.5-0.6 parts of glycerol, 0.3-0.5 parts of peppermint oil and 0.1-0.3 parts of oil-soluble azone.

2. Toad skin ointment according to claim 1, characterized in that, The toad skin extract is an extract of the dried whole skin of Bufo bufo gargarizans.

3. Toad skin ointment according to claim 2, characterized in that, The preparation method of the toad skin extract comprises the following steps: mixing the toad skin with ethanol, heating and refluxing for extraction, centrifuging the extract to obtain the supernatant, concentrating until there is no alcohol taste, and then freeze-drying to obtain toad skin freeze-dried powder.

4. Toad skin ointment according to claim 3, characterized in that, The solid-liquid ratio of the toad skin to ethanol is 1g:15-25mL; the ethanol is a 70-90% ethanol solution; the heating reflux extraction time is 2-3h; and the heating reflux extraction times is 1-2 times.

5. Toad skin ointment according to claim 3, characterized in that, The centrifugal gravity acceleration is 1700-1800×g, the time is 15-20 min; the concentration temperature is 50-60° C.

6. The toad skin ointment according to claim 3, characterized in that The freeze drying temperature is -10 to -20°C, the vacuum degree is 150 to 300 Pa, and the time is 12 to 24 hours.

7. The method for preparing the toad skin ointment according to any one of claims 1 to 6, characterized in that: The following steps are involved: Vaseline, lanolin and liquid paraffin are dissolved and mixed, glycerin and toad skin extract are added, mixed evenly, peppermint oil and oil-soluble azone are added, mixed evenly, and cooled to obtain toad skin ointment.

8. The preparation method according to claim 7, characterized in that: The dissolving step is to dissolve by heating in a water bath at 60-70°C.

9. Use of the toad skin ointment according to any one of claims 1 to 6 or the preparation method according to any one of claims 7 to 8 in the preparation of anti-edema and analgesic drugs.

10. Use of the toad skin ointment according to any one of claims 1 to 6 or the preparation method according to any one of claims 7 to 8 in the preparation of anti-inflammatory drugs.

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